In this report, we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-IC(ML)) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro. The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and than for an additional week under conditions permissive for B-lymphopoiesis. All of the clonogenic pre-B progenitors (colony-forming unit [CFU] pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for ≥7 days was found to irreversibly terminate CFU-pre-B production and, in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in >70% of cultures 1 week after the switch), the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells, regardless of the purity of the input population. Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-IC(ML) in normal adult mouse marrow to be 1 per 5 x 105 cells with an enrichment of ~500-fold in the Sca-1+Lin-WGA+ fraction, as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC. LTC-IC(ML) also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC, thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S. The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro, without the need for their prior purification, should facilitate studies of totipotent hematopoietic stem cell regulation.
|Original language||English (US)|
|Number of pages||9|
|State||Published - 1995|
ASJC Scopus subject areas
- Cell Biology